Spectrophotometry is used to determine whether the measured substancewavelengthOptical at or within a certain wavelength rangeAbsorbance, for thematerialQualitative andquantitative analysisMethod.It has the advantages of high sensitivity, simple operation and fast speed. It is the most commonly used experimental method in biochemical experiments.Many substances are determined bySpectrophotometry。staySpectrophotometer, continuously irradiate light of different wavelengths to a certainconcentrationOfsampleSolution, you can getwavelengthCorrespondingAbsorption strength。
staySpectrophotometerWhen light of different wavelengths is continuously irradiated to a certain concentration of sample solution, the absorption intensity corresponding to different wavelength can be obtained.If the wavelength (λ) is taken as the abscissa and the absorption intensity (A) as the ordinate, theAbsorption spectrum curve。This curve is used for qualitative and quantitative analysis of substances, which is called spectrophotometry or absorption spectrometry.The method of measuring colorless substances with ultraviolet light source is calledUltraviolet spectrophotometry；The method of measuring colored substances with visible light source is called visible light photometry.Like colorimetry, they are based on Lambert Bee law.[1]
The above ultraviolet and visible light areas are commonly used.However, the application light area of spectrophotometry includes ultraviolet light area, visible light area and infrared light area.Wavelength range:
1. 200~400nm ultraviolet light zone
2. 400 ～ 760nm visible light area
3. 2.5 ～ 25 μ m (4000cm by wave number-1～400cm-1)The infrared light area of.[2]
Fundamentals
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Selective absorption
The substance interacts with light and has the characteristic of selective absorption.The color of a colored substance is produced by its interaction with light.That is, the color of the colored solution is due to the selective absorption of light by the substances in the solution.Because different substances have different molecular structures and different absorption capacities for light of different wavelengths, the structural groups with characteristic structures have the maximum received wavelength to select the absorption characteristics, form the maximum absorption peak, and produce a unique absorption spectrum.Even the same substance has different light absorption due to its different content.The method of using the specific absorption spectrum of a substance to identify the existence of a substance (qualitative analysis), or using the absorption degree of a substance to a certain wavelength of light to determine the content of a substance (quantitative analysis), is called spectrophotometry.[2]
Quantitative basis
Lambert - Beer law is the basic principle of quantitative analysis by spectrophotometry.When a beam of monochromatic light (light of one color) passes through a uniform solution, part is absorbed and part is transmitted.Set the intensity of incident light asIzero, the transmitted light intensity is I, then I/IzeroIs the light transmittance, which is represented by T.Percentage transmittance is T%=（I/Izero）x100%
The research shows that the absorption degree of solution to light isabsorbance(A) (also called extinction E, oroptical densityD) AndTransmittance(T) Negative logarithmic relationship, i.e. A=- lgT
Lambert's law: The absorbance degree (A) of colored solution is proportional to the liquid layer thickness (optical path) b.When the concentration of the solution is constant, the greater the thickness of the solution layer, the greater the light absorption A value, and the smaller the light transmittance.
That is, A=ab
Beer's law: The absorbance degree (A) of colored solution is proportional to its concentration (absorbance quality point) C.That is, when the thickness of the liquid layer of the solution is constant, the greater the concentration of the solution, the greater the absorption of light, and the smaller the transmittance.
That is, A=ac
The combination of the above two expressions can be expressed as follows: A=- lgT=abc
That is, A=abc.
The above formula islambert-beer law , which means that when a beam of monochromatic light passes through a uniform solution, the absorbance of the solution to monochromatic light is proportional to the product of solution concentration and liquid layer thickness.[2-3]
A=abc,Light absorption coefficienta，Characterize the sensitivity of light absorbing materials.The greater the value of a, the higher the sensitivity.If the unit of concentration is the amount concentration of the substance (unit: mol/L), the absorption coefficient a can be written as ε, and ε is calledMolar absorptivity。
It can be seen from the above formula that when the thickness of the solution layer b and the absorption coefficient a are fixed, the absorbance A is equal to the concentration of the solutionlinear relationship。In quantitative analysis, it is first necessary to determine the absorption of the solution to light of different wavelengths (absorption spectrum), determine the maximum absorption wavelength, and then use the light of this wavelength as the light source (monochromatic light) to determine the absorbance A. This is the primary condition for Lambert Beer's law to be used for quantitative analysis.[2]
The basic components of various spectrophotometers are: light source system, spectrophotometer system, absorption system and detection system.[2]
Instrument accuracy
In order to ensure the precision and accuracy of measurement, all instruments shall be calibrated regularly according to the national metrological verification regulations.
quantitative method
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Quantitative determination of substances by spectrophotometry mainly includes the following methods:
1. Standard tube method
Measure the A value of the solution to be tested and the standard solution of known concentration under the same conditions, and then calculate the content of the substance in the solution to be tested according to the following formula.
First, prepare a series of standard solutions with the concentration from small to large, and measure their A values respectively. With A value as the abscissa and concentration as the ordinate, make the standard curve (A~c working curve), and then get the regression equation of the standard curve.When determining the solution to be tested, the operating conditions should be the same as when making the standard curve, so that the corresponding concentration of the sample can be found (obtained) from the standard curve with the A value of the solution to be tested.
3. Absorption coefficient method
When the concentration of a substance solution is 1mol/L and the thickness is 1cm, the absorbance of the solution to a certain wavelength is called the molar absorbance coefficient of the substance, expressed in ε.The ε value can be measured by experiment or found in the manual.For example, the ε value of ferrous nitrogen phenanthrene complex is equal to 11000 L/cm · mol
If the ε value of a substance is known, the sample concentration can be obtained by measuring its A value according to the following formula: c=A/ε.
